Malformations of cortical development due to disorders of neuronal migration are increasingly recognized as a common cause of epilepsy, mental retardation and cerebral palsy. The doublecortin (DCX) gene is critical for neuronal migration in humans, as mutations result in X-linked lissencephaly in males and subcortical band heterotopia in females, producing severe neurocognitive deficits. We identified the DCX gene, and found mutations in patients with this condition. We identified its role as a microtubule (MT)-associated protein and its involvement in several signaling pathways through phosphorylation-dependent mechanisms. We also identified a potential role for Dcx in coupling the nucleus to the centrosome in a microtubule-dependent fashion during the nuclear translocation phase of migration. Dcx is part of a gene family also containing Dck1 and Dck2, each encoding a strongly brain-expressed protein with a closely matching Dcx domain and a kinase domain. The overall goal of this renewal application is to elucidate the molecular and cellular mechanisms of the Dcx gene family in neuronal migration and brain function. We will utilize knockout and transgenic reporter mice combined with advanced live-cell imaging capabilities and in vivo analysis that will synergize to provide a powerful approach address this goal. Aim 1. Test the degree of functional redundancy of Dcx homologues Dck1 and Dck2 in neuronal migration and brain development. The function of Dcx in migration may be redundant with the Dck1 and Dck2 genes in mouse. We will analyze the degree of functional redundancy through the analysis of phenotype of Dck1 and Dck2 single as well as double and triple knockout mice and compare these results with siRNA-mediated gene knockdown approaches. Aim 2. Test for defects in MT stabilization and nuclear-centrosomal coupling in Dcx-family gene inactivation. Utilizing the approaches from Aim 1 and advanced live cell imaging techniques, we will test whether the Dcx gene family is required for MT-dependent nuclear movement in neuronal migration. Aim 3. Test for phosphorylation and phosphatase-dependent regulation of the MT effects of the Dcx gene family. Our previous data has indicated strong negative-regulation of Dcx function through phosphorylation. We now have genetic and biochemical data that actin-linked protein-phosphatase I and MT-linked Dck1/2 provide additional levels of phosphorylation-dependent regulation. We will test the specificity of these interactions using the reagents generated here and test their role in integrating the microtubule and actin cytoskeletons required for stabilization of neuronal growth cones. Lay Summary: Mutations in doublecortin lead to severe neurological disorders in humans due to altered brain development through unknown mechanisms. This study seeks to identify the function the family of doublecortin genes using advanced molecular and cellular approaches. [unreadable] [unreadable]